The bacterial reverse mutation test (Ames test) was performed using the preincubation
method as described byMortelmans and Zeiger
(2000) , with slight alterations. Briefly, SalmonellaTyphimurium TA98 (Trinova, Gießen, Germany) was cultivated in nutrient broth (nutrient
broth no. 2; Oxoid, Wesel, Germany) overnight and then used at an OD of 0.9–1.0 in the
test. Bacteria were thereby incubated with top agar (Kobe I agar; Carl Roth), S9
originating from rat liver (Trinova) and sodium phosphate buffer (2.8 g/l
NaH2PO4·H2O, 12.8 g/l
Na2HPO4·2H2O, 2.7 g/l KCl, 1.8 g/l MgCl, 3.5 g/l
NADP, 1.6 g/l glucose-6-phosphate, pH = 7.4) for 20 min at 37 °C in 15 ml reaction
tubes (CELLSTAR polypropylene tubes; Greiner Bio-One; Frickenhausen, Germany). This
mixture was then poured into petri dishes (Sarstedt, Nümbrecht, Germany) containing
base agar (Kobe I agar, Carl Roth). Finally, the plates were incubated for 48 h at
37 °C and the colonies on each plate were counted. To test the stability of MelQx-M1
under the assay conditions, buffer containing 10% of rat liver S9 and 1 µM MelQx-M1
were incubated for 30 min at 37 °C, before the mixture was centrifuged and the
supernatant analyzed in the same manner as the samples described in
LC-ESI-MS2 section.
method as described by
(2000)
broth no. 2; Oxoid, Wesel, Germany) overnight and then used at an OD of 0.9–1.0 in the
test. Bacteria were thereby incubated with top agar (Kobe I agar; Carl Roth), S9
originating from rat liver (Trinova) and sodium phosphate buffer (2.8 g/l
NaH2PO4·H2O, 12.8 g/l
Na2HPO4·2H2O, 2.7 g/l KCl, 1.8 g/l MgCl, 3.5 g/l
NADP, 1.6 g/l glucose-6-phosphate, pH = 7.4) for 20 min at 37 °C in 15 ml reaction
tubes (CELLSTAR polypropylene tubes; Greiner Bio-One; Frickenhausen, Germany). This
mixture was then poured into petri dishes (Sarstedt, Nümbrecht, Germany) containing
base agar (Kobe I agar, Carl Roth). Finally, the plates were incubated for 48 h at
37 °C and the colonies on each plate were counted. To test the stability of MelQx-M1
under the assay conditions, buffer containing 10% of rat liver S9 and 1 µM MelQx-M1
were incubated for 30 min at 37 °C, before the mixture was centrifuged and the
supernatant analyzed in the same manner as the samples described in
LC-ESI-MS2 section.